Abstract The innate immune system forms the first line of specific host defense against invading microbial pathogens. Integral to the innate immune response are Pattern Recognition Receptors (PRRs), which recognize conserved determinants expressed by a broad range of pathogenic microorganisms. Among these are the Toll-like receptors, which promote inflammatory signaling and phagocyte recruitment in response to pathogen recognition, and several families of phagocytic PRRs that mediate phagocytic clearance of microbes from infected tissues. Each receptor family contains multiple members that recognize a unique spectrum of microbial components, and collectively they provide protection against a wide range of infectious organisms. We have identified a novel pattern recognition receptor, BAI1, that specifically recognizes the surface lipopolysaccharide (LPS) of Gram-negative bacteria. BAI1 is a member of a poorly understood family of G- protein coupled receptors (GPCRs), the so-called Adhesion GPCRs. Binding of bacteria to BAI1 triggers their rapid internalization and killing by host phagocytes, through a mechanism involving activation of the small GTPase Rac by the Elmo/Dock GEF complex, which interacts directly with BAI1. Whether and how signaling through heterotrimeric G proteins is integrated into this process is not known, and will be the focus of Aim 1. An emerging theme in innate immunity is that pattern recognition receptors often cooperate with each other to enhance downstream signaling responses. Our published and preliminary data indicate that BAI1 acts in tandem with the Toll-like receptor TLR4 to promote the production of reactive oxygen species (ROS) by phagocytic cells, and the synthesis of a subset of inflammatory cytokines/chemokines/interferons whose expression is mediated by the transcription factor IRF3. In Aim 2 we will determine how signals derived from BAI1 influence TLR4-mediated signaling to drive ROS production and the transcription of pro-inflammatory genes. The ultimate goal of the proposed research is to define the role of BAI1 in host defense against bacterial infection. In Aim 3, we will characterize the ability of BAI1-deficient macrophages and neutrophils to clear infection in a mouse model of Gram-negative peritonitis. The ability of BAI1 to drive ROS production and cytokine/interferon synthesis in specific myeloid cell subsets during infection will also be determined. Together these studies will provide unique biochemical and physiological insights into a new class of pattern recognition receptor with important functions in the innate immune response to bacterial pathogens. Moreover, BAI1 is one of the few Adhesion GPCRs with known physiological ligands, and dissection of its signaling properties will reveal new insights into this poorly understood class of receptors.